The HaDREB2 transcription factor enhances basal thermotolerance and longevity of seeds through functional interaction with HaHSFA9

Abstract

Background: Transcription factor HaDREB2 was identified in sunflower (Helianthus annuus L.) as a drought-responsive element-binding factor 2 (DREB2) with unique properties. HaDREB2 and the sunflower Heat Shock Factor A9 (HaHSFA9) co-activated the Hahsp17.6G1 promoter in sunflower embryos. Both factors could be involved in transcriptional co-activation of additional small heat stress protein (sHSP) promoters, and thus contribute to the HaHSFA9-mediated enhancement of longevity and basal thermotolerance of seeds.

Results: We found that overexpression of HaDREB2 in seeds did not enhance longevity. This was deduced from assays of basal thermotolerance and controlled seed-deterioration, which were performed with transgenic tobacco. Furthermore, the constitutive overexpression of HaDREB2 did not increase thermotolerance in seedlings or result in the accumulation of HSPs at normal growth temperatures. In contrast, when HaDREB2 and HaHSFA9 were conjointly overexpressed in seeds, we observed positive effects on seed longevity, beyond those observed with overexpression of HaHSFA9 alone. Such additional effects are accompanied by a subtle enhancement of the accumulation of subsets of sHSPs belonging to the CI and CII cytosolic classes.

Conclusion: Our results reveal the functional interdependency of HaDREB2 and HaHSFA9 in seeds. HaDREB2 differs from other previously characterized DREB2 factors in plants in terms of its unique functional interaction with the seed-specific HaHSFA9 factor. No functional interaction between HaDREB2 and HaHSFA9 was observed when both factors were conjointly overexpressed in vegetative tissues. We therefore suggest that additional, seed-specific factors, or protein modifications, could be required for the functional interaction between HaDREB2 and HaHSFA9.

Figure 1

Unaltered vegetative thermotolerance and HSP accumulation in heterozygous 35S:DR2 lines. A. Seedlings from the 35S:DR2 lines do not survive direct exposure to 48°C for 2.5 h (48°C). However the 35S:DR2 seedlings survive the same heat stress treatment following heat acclimation [48°C (HA)]. Representative pictures of the 35S:DR2#1 line are shown here. B. Western blot showing accumulation of HA-tagged HaDREB2 (HA-DR2) in the 35S:DR2 lines. Detection with antibodies against the HA tag. The accumulation of different HSPs was detected in heat stressed, control plants (HS) but not in unstressed 35S:DR2 plants from the analyzed lines. The HSP-specific antibodies used for immunodetection are indicated on the right. Molecular mass markers (in kDa) are indicated on the left.

Figure 2

Improved thermotolerance and enhanced resistance to CDT in seeds from the DS10:A9/DR2 heterozygous lines. Segregation of hygromycin resistance (Hyg^R) was analyzed in germinating seeds before and after the indicated treatment. BTA, basal thermotolerance assays; CDT, controlled deterioration treatments. Experiments were performed at least in triplicate for each line. A. – Combined results from five lines with the DS10:A9#6-7 homozygous background (A9#6-7/DR2), four lines with the DS10:A9#14-5 background (A9#14-5/DR2), and eight DS10:DR2 lines (DR2) are shown. Segregation was analyzed five (in DS10:A9/DR2 lines) or eight days (in DS10:DR2 lines) after BTA. Segregation was analyzed five days after CDT. The expected segregation ratio for antibiotic resistance is indicated with a thick line (marking 3 Hyg^R: 1 Hyg^S). Asterisks indicate differences that were found to be statistically significant (P < 0.05). We show representative pictures of segregation of hygromycin resistance for the DS10:A9#6-7/DR2#22 line after BTA (B) and before BTA (C). Arrows in (B) indicate the few Hyg^S seedlings that survived BTA. Scale bars, 8 mm.

Figure 3

The combined DS10:DR2 and DS10:A9 transgenes in homozygosis enhance seed survival after BTA. A. Germination after BTA of double-homozygous seeds (A9/DR2) compared to that of seeds from sibling lines without DR2 (A9). We show average germination at different time after BTA for 3 to 4 independent experiments performed with two pairs of A9/DR2 lines (A9#14-5/DR2#5-7 and A9#14-5/DR2#23-5) compared to their respective sibling lines without DR2 (A9#14-5/#5-4 and A9#14-5/#23-6). Also shown are pictures of representative results of individual experimental samples showing germination 15 d after BTA: B. A9#14-5/DR2#5-7 (A9+DR2). C. A9#14-5/#5-4 (A9). Scale bars, 10 mm.

Figure 4

Specific enhancement of the accumulation of some sHSPs in seeds of DS10:A9/DR2 lines. Comparison of the accumulation patterns of sHSP-CI and sHSP-CII. A representative, double-homozygous DS10:A9/DR2 line (A9/DR2) is compared to its sibling DS10:A9 line (A9). Asterisks mark the polypeptides that consistently showed higher accumulation in the DS10:A9/DR2 lines. Molecular mass markers (in kDa) are indicated on the left. The pH range for isoelectric focusing (IEF) is indicated (bottom).

Figure 5

The 35S:DR2 transgene when combined with 35S:A9 does not further enhance tolerance to severe dehydration. Average survival of whole seedlings after DT2 assays (top). Average water potential (Φ) reached in the different DT2 assays (bottom). Data were obtained from two 35S:A9/DR2 lines (A9/DR2), 35S:A9#2-18/DR2#1-6, 35S:A9#12-3/DR2#22-3, and the corresponding sibling 35S:A9 lines (A9). Each pair of sibling lines represents the genetic background of the previously analyzed parental 35S:A9 lines [4]. Data were obtained from 3 to 14 experimental repeats per line and average water potential condition.